Adenovirus‑mediated knockdown of activin A receptor type 2A attenuates immune‑induced hepatic fibrosis in mice and inhibits interleukin‑17‑induced activation of primary hepatic stellate cells

Zhang,Hongjun; Ju,Baoling; Nie,Ying; Song,Baohui; Xu,Yuanhong; Gao,Ping

doi:10.3892/ijmm.2018.3600

Adenovirus‑mediated knockdown of activin A receptor type 2A attenuates immune‑induced hepatic fibrosis in mice and inhibits interleukin‑17‑induced activation of primary hepatic stellate cells

Authors:
- Hongjun Zhang
- Baoling Ju
- Ying Nie
- Baohui Song
- Yuanhong Xu
- Ping Gao
View Affiliations

Affiliations: Department of Immunology, Mudanjiang Medical University, Mudanjiang, Heilongjiang 157011, P.R. China, Department of Gastroenterology, Mudanjiang Forestry Central Hospital, Mudanjiang, Heilongjiang 157000, P.R. China
Published online on: March 29, 2018 https://doi.org/10.3892/ijmm.2018.3600
Pages: 279-289
Copyright: © Zhang et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )

Metrics: Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )

Cited By (CrossRef): 0 citations Loading Articles...

Abstract

Fibrosis induces a progressive loss of liver function, thus leading to organ failure. Activins are secreted proteins that belong to the transforming growth factor (TGF)‑β superfamily, which initiate signaling by binding to their two type II receptors: Activin A receptor type 2A (ACVR2A) and activin A receptor type 2B. Previous studies that have explored the mechanisms underlying immune‑induced hepatic fibrosis have mainly focused on TGF‑β signaling, not activin signaling. To investigate the role of the activin pathway in this disease, adenovirus particles containing short hairpin (sh)RNA targeting ACVR2A mRNA (Ad‑ACVR2A shRNA) were administered to mice, which were chronically treated with concanavalin A (Con A). The pathological changes in the liver were evaluated with hematoxylin/eosin staining, Masson trichrome staining and immunohistochemical assay. The results detected an increase in serum activin A and liver ACVR2A in Con A‑treated animals. Conversely, liver function was partially restored and fibrotic injury was attenuated when activin signaling was blocked. In addition, the activation of hepatic stellate cells (HSCs) in response to Con A was suppressed by Ad‑ACVR2A shRNA, as evidenced by decreased α‑smooth muscle actin, and type I and IV collagen expression. Furthermore, primary mouse HSCs (mHSCs) were activated when exposed to interleukin (IL)‑17A or IL‑17F, which are two major cytokines produced by cluster of differentiation 4+ T helper 17 cells. The levels of activin A, type I and IV collagen were determined with ELISA kits and the expression of fibrotic molecules was determined with western blot analysis. Conversely, blocking activin/ACVR2A impaired the potency of HSCs to produce collagens in response to IL‑17s. In addition, C terminus phosphorylation of Smad2 on Ser465 and Ser467, induced by either Con A in the liver or by IL‑17s in mHSCs, was partly inhibited when activin A/ACVR2A signaling was suppressed. Collectively, the present study demonstrated an involvement of activated activin A/ACVR2A/Smad2 signaling in immune‑induced hepatic fibrosis.

View Figures

View References

1	Pellicoro A, Ramachandran P, Iredale JP and Fallowfield JA: Liver fibrosis and repair: Immune regulation of wound healing in a solid organ. Nat Rev Immunol. 14:181–194. 2014. View Article : Google Scholar : PubMed/NCBI
2	Nanthakumar CB, Hatley RJ, Lemma S, Gauldie J, Marshall RP and Macdonald SJ: Dissecting fibrosis: Therapeutic insights from the small-molecule toolbox. Nat Rev Drug Discov. 14:693–720. 2015. View Article : Google Scholar : PubMed/NCBI
3	Lee YA, Wallace MC and Friedman SL: Pathobiology of liver fibrosis: A translational success story. Gut. 64:830–841. 2015. View Article : Google Scholar : PubMed/NCBI
4	Mederacke I, Hsu CC, Troeger JS, Huebener P, Mu X, Dapito DH, Pradere JP and Schwabe RF: Fate tracing reveals hepatic stellate cells as dominant contributors to liver fibrosis independent of its aetiology. Nat Commun. 4:28232013. View Article : Google Scholar : PubMed/NCBI
5	Chen RJ, Wu HH and Wang YJ: Strategies to prevent and reverse liver fibrosis in humans and laboratory animals. Arch Toxicol. 89:1727–1750. 2015. View Article : Google Scholar : PubMed/NCBI
6	Seki E and Schwabe RF: Hepatic inflammation and fibrosis: Functional links and key pathways. Hepatology. 61:1066–1079. 2015. View Article : Google Scholar :
7	Bissell DM, Roulot D and George J: Transforming growth factor beta and the liver. Hepatology. 34:859–867. 2001. View Article : Google Scholar : PubMed/NCBI
8	Nakamura T, Sakata R, Ueno T, Sata M and Ueno H: Inhibition of transforming growth factor beta prevents progression of liver fibrosis and enhances hepatocyte regeneration in dimethylnitrosamine-treated rats. Hepatology. 32:247–255. 2000. View Article : Google Scholar : PubMed/NCBI
9	Qi Z, Atsuchi N, Ooshima A, Takeshita A and Ueno H: Blockade of type beta transforming growth factor signaling prevents liver fibrosis and dysfunction in the rat. Proc Natl Acad Sci USA. 96:2345–2349. 1999. View Article : Google Scholar : PubMed/NCBI
10	Weiss A and Attisano L: The TGFbeta superfamily signaling pathway. Wiley Interdiscip Rev Dev Biol. 2:47–63. 2013. View Article : Google Scholar : PubMed/NCBI
11	Kreidl E, Oztürk D, Metzner T, Berger W and Grusch M: Activins and follistatins: Emerging roles in liver physiology and cancer. World J Hepatol. 1:17–27. 2009. View Article : Google Scholar : PubMed/NCBI
12	Sugiyama M, Ichida T, Sato T, Ishikawa T, Matsuda Y and Asakura H: Expression of activin A is increased in cirrhotic and fibrotic rat livers. Gastroenterology. 114:550–558. 1998. View Article : Google Scholar : PubMed/NCBI
13	Gold EJ, Francis RJ, Zimmermann A, Mellor SL, Cranfield M, Risbridger GP, Groome NP, Wheatley AM and Fleming JS: Changes in activin and activin receptor subunit expression in rat liver during the development of CCl₄-induced cirrhosis. Mol Cell Endocrinol. 201:143–153. 2003. View Article : Google Scholar : PubMed/NCBI
14	Fujita T, Soontrapa K, Ito Y, Iwaisako K, Moniaga CS, Asagiri M, Majima M and Narumiya S: Hepatic stellate cells relay inflammation signaling from sinusoids to parenchyma in mouse models of immune-mediated hepatitis. Hepatology. 63:1325–1339. 2016. View Article : Google Scholar
15	Louis H, Le Moine A, Quertinmont E, Peny MO, Geerts A, Goldman M, Le Moine O and Devière J: Repeated concanavalin A challenge in mice induces an interleukin 10-producing phenotype and liver fibrosis. Hepatology. 31:381–390. 2000. View Article : Google Scholar : PubMed/NCBI
16	Tu CT, Li J, Wang FP, Li L, Wang JY and Jiang W: Glycyrrhizin regulates CD4+ T cell response during liver fibrogenesis via JNK, ERK and PI3K/AKT pathway. Int Immunopharmacol. 14:410–421. 2012. View Article : Google Scholar : PubMed/NCBI
17	Meng F, Wang K, Aoyama T, Grivennikov SI, Paik Y, Scholten D, Cong M, Iwaisako K, Liu X, Zhang M, et al: Interleukin-17 signaling in inflammatory, Kupffer cells, and hepatic stellate cells exacerbates liver fibrosis in mice. Gastroenterology. 143:765–76. e1–3. 2012. View Article : Google Scholar : PubMed/NCBI
18	Song E, Lee SK, Wang J, Ince N, Ouyang N, Min J, Chen J, Shankar P and Lieberman J: RNA interference targeting Fas protects mice from fulminant hepatitis. Nat Med. 9:347–351. 2003. View Article : Google Scholar : PubMed/NCBI
19	Yu PB, Beppu H, Kawai N, Li E and Bloch KD: Bone morphogenetic protein (BMP) type II receptor deletion reveals BMP ligand-specific gain of signaling in pulmonary artery smooth muscle cells. J Biol Chem. 280:24443–24450. 2005. View Article : Google Scholar : PubMed/NCBI
20	Xu WH, Hu HG, Tian Y, Wang SZ, Li J, Li JZ, Deng X, Qian H, Qiu L, Hu ZL, et al: Bioactive compound reveals a novel function for ribosomal protein S5 in hepatic stellate cell activation and hepatic fibrosis. Hepatology. 60:648–660. 2014. View Article : Google Scholar : PubMed/NCBI
21	Tan Z, Qian X, Jiang R, Liu Q, Wang Y, Chen C, Wang X, Ryffel B and Sun B: IL-17A plays a critical role in the pathogenesis of liver fibrosis through hepatic stellate cell activation. J Immunol. 191:1835–1844. 2013. View Article : Google Scholar : PubMed/NCBI
22	Li Y, Kim BG, Qian S, Letterio JJ, Fung JJ, Lu L and Lin F: Hepatic stellate cells inhibit T cells through active TGF-β1 from a cell surface-bound latent TGF-β1/GARP complex. J Immunol. 195:2648–2656. 2015. View Article : Google Scholar : PubMed/NCBI
23	Yu MC, Chen CH, Liang X, Wang L, Gandhi CR, Fung JJ, Lu L and Qian S: Inhibition of T-cell responses by hepatic stellate cells via B7-H1-mediated T-cell apoptosis in mice. Hepatology. 40:1312–1321. 2004. View Article : Google Scholar : PubMed/NCBI
24	Weiskirchen S, Tag CG, Sauer-Lehnen S, Tacke F and Weiskirchen R: Isolation and culture of primary murine hepatic stellate cells. Methods Mol Biol. 1627:165–191. 2017. View Article : Google Scholar : PubMed/NCBI
25	Livak KJ and Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 25:402–408. 2001. View Article : Google Scholar
26	Chang J, Lan T, Li C, Ji X, Zheng L, Gou H, Ou Y, Wu T, Qi C, Zhang Q, et al: Activation of Slit2-Robo1 signaling promotes liver fibrosis. J Hepatol. 63:1413–1420. 2015. View Article : Google Scholar : PubMed/NCBI
27	Patella S, Phillips DJ, Tchongue J, de Kretser DM and Sievert W: Follistatin attenuates early liver fibrosis: Effects on hepatic stellate cell activation and hepatocyte apoptosis. Am J Physiol Gastrointest Liver Physiol. 290:G137–G144. 2006. View Article : Google Scholar
28	Patella S, Phillips DJ, de Kretser DM, Evans LW, Groome NP and Sievert W: Characterization of serum activin-A and follistatin and their relation to virological and histological determinants in chronic viral hepatitis. J Hepatol. 34:576–583. 2001. View Article : Google Scholar : PubMed/NCBI
29	Wang DH, Wang YN, Ge JY, Liu HY, Zhang HJ, Qi Y, Liu ZH and Cui XL: Role of activin A in carbon tetrachloride-induced acute liver injury. World J Gastroenterol. 19:3802–3809. 2013. View Article : Google Scholar : PubMed/NCBI
30	George J, Roulot D, Koteliansky VE and Bissell DM: In vivo inhibition of rat stellate cell activation by soluble transforming growth factor beta type II receptor: A potential new therapy for hepatic fibrosis. Proc Natl Acad Sci USA. 96:12719–12724. 1999. View Article : Google Scholar : PubMed/NCBI
31	Fenoglio D, Bernuzzi F, Battaglia F, Parodi A, Kalli F, Negrini S, De Palma R, Invernizzi P and Filaci G: Th17 and regulatory T lymphocytes in primary biliary cirrhosis and systemic sclerosis as models of autoimmune fibrotic diseases. Autoimmun Rev. 12:300–304. 2012. View Article : Google Scholar : PubMed/NCBI
32	Chen Y, Peng H, Chen Y, Wei H, Sun R and Tian Z: CD49a promotes T-cell-mediated hepatitis by driving T helper 1 cytokine and interleukin-17 production. Immunology. 141:388–400. 2014. View Article : Google Scholar :
33	Kolls JK and Lindén A: Interleukin-17 family members and inflammation. Immunity. 21:467–476. 2004. View Article : Google Scholar : PubMed/NCBI
34	Wang Q, Zhou J, Zhang B, Tian Z, Tang J, Zheng Y, Huang Z, Tian Y, Jia Z, Tang Y, et al: Hepatitis B virus induces IL-23 production in antigen presenting cells and causes liver damage via the IL-23/IL-17 axis. PLoS Pathog. 9:e10034102013. View Article : Google Scholar : PubMed/NCBI
35	Seeger P, Bosisio D, Parolini S, Badolato R, Gismondi A, Santoni A and Sozzani S: Activin A as a mediator of NK-dendritic cell functional interactions. J Immunol. 192:1241–1248. 2014. View Article : Google Scholar : PubMed/NCBI
36	González-Domínguez É, Domínguez-Soto Á, Nieto C, Flores-Sevilla JL, Pacheco-Blanco M, Campos-Peña V, Meraz-Ríos MA, Vega MA, Corbí ÁL and Sánchez-Torres C: Atypical activin A and IL-10 production impairs human CD16+ monocyte differentiation into anti-inflammatory macrophages. J Immunol. 196:1327–1337. 2016. View Article : Google Scholar : PubMed/NCBI
37	Wada W, Kuwano H, Hasegawa Y and Kojima I: The dependence of transforming growth factor-beta-induced collagen production on autocrine factor activin A in hepatic stellate cells. Endocrinology. 145:2753–2759. 2004. View Article : Google Scholar : PubMed/NCBI
38	Macias MJ, Martin-Malpartida P and Massagué J: Structural determinants of Smad function in TGF-β signaling. Trends Biochem Sci. 40:296–308. 2015. View Article : Google Scholar : PubMed/NCBI
39	Chen L, Zhang W, Liang HF, Zhou QF, Ding ZY, Yang HQ, Liu WB, Wu YH, Man Q, Zhang BX, et al: Activin A induces growth arrest through a SMAD- dependent pathway in hepatic progenitor cells. Cell Commun Signal. 12:182014. View Article : Google Scholar : PubMed/NCBI
40	Yoshida K, Murata M, Yamaguchi T, Matsuzaki K and Okazaki K: Reversible human TGF-β signal shifting between tumor suppression and fibro-carcinogenesis: Implications of Smad phospho-isoforms for hepatic epithelial-mesenchymal transitions. J Clin Med. 5:52016. View Article : Google Scholar
41	Souchelnytskyi S, Tamaki K, Engström U, Wernstedt C, ten Dijke P and Heldin CH: Phosphorylation of Ser465 and Ser467 in the C terminus of Smad2 mediates interaction with Smad4 and is required for transforming growth factor-beta signaling. J Biol Chem. 272:28107–28115. 1997. View Article : Google Scholar : PubMed/NCBI
42	Wynn TA: Fibrotic disease and the T(H)1/T(H)2 paradigm. Nat Rev Immunol. 4:583–594. 2004. View Article : Google Scholar
43	Veldhoen M, Hocking RJ, Atkins CJ, Locksley RM and Stockinger B: TGFbeta in the context of an inflammatory cytokine milieu supports de novo differentiation of IL-17-producing T cells. Immunity. 24:179–189. 2006. View Article : Google Scholar : PubMed/NCBI
44	Kang JO, Lee JB and Chang J: Cholera toxin promotes Th17 cell differentiation by modulating expression of polarizing cytokines and the antigen-presenting potential of dendritic cells. PLoS One. 11:e01570152016. View Article : Google Scholar : PubMed/NCBI
45	Ihn HJ, Kim DH, Oh SS, Moon C, Chung JW, Song H and Kim KD: Identification of Acvr2a as a Th17 cell-specific gene induced during Th17 differentiation. Biosci Biotechnol Biochem. 75:2138–2141. 2011. View Article : Google Scholar : PubMed/NCBI